Substituent effects upon the catalytic activity of aromatic cyclic seleninate esters and spirodioxyselenuranes that act as glutathione peroxidase mimetics

Article abstract of DOI:10.1021/jo800381s

(Chemical Equation Presented) Substituent effects were studied in a series of aromatic cyclic seleninate esters and spirodioxyselenuranes that function as mimetics of the antioxidant selenoenzyme glutathione peroxidase. The methoxy-substituted selenurane proved the most efficacious catalyst for the reduction of hydrogen peroxide with benzyl thiol, and the reaction rates were enhanced for both classes by electron-donating substituents. Hammett plots indicated ρ = -0.45 and -3.1 for the seleninates and selenuranes, respectively, suggesting that oxidation at Se is the rate-determining step in their catalytic cycles.

Full text of DOI:10.1021/jo800381s

SCHEME 1
Substituent Effects upon the Catalytic Activity of
Aromatic Cyclic Seleninate Esters and
Spirodioxyselenuranes That Act as Glutathione
Peroxidase Mimetics
David J. Press, Eric A. Mercier, Dusˇan Kuzma, and
Thomas G. Back*
Department of Chemistry, UniVersity of Calgary, Calgary,
Alberta, Canada T2N 1N4
tgback@ucalgary.ca
species. The selenoenzyme glutathione peroxidase (GPx) per-
forms a vital role in this regard by catalyzing the reduction of
peroxides with glutathione (GSH), a tripeptide thiol that is
abundant in the cells of higher organisms and serves as a
stoichiometric reductant in this process. The catalytic cycle of
the enzyme has been established and is shown in Scheme 1.5
The selenol moiety (EnzSeH) associated with each selenocys-
teine residue in the enzyme6 reduces peroxides readily and is
itself oxidized to the corresponding selenenic acid (EnzSeOH).
The latter then reacts with two molecules of GSH to regenerate
the selenol via the selenenyl sulfide EnzSeSG, along with a
stoichiometric amount of glutathione disulfide (GSSG). At high
peroxide concentrations, the corresponding seleninic acid in-
termediate (EnzSeO2H) may also play a role in the process.
ReceiVed February 15, 2008
The design, synthesis, and evaluation for GPx-like activity
of small-molecule selenium compounds, as well as of selenium-
containing macromolecules, have been the subject of consider-
able investigation.7 Ebselen (1)8,9 and ALT 2074 (2)10 are
undergoing clinical trials as antioxidants, particularly for
counteracting high levels of oxidative stress associated with
strokes and related cardiovascular conditions. We recently
discovered two types of compounds that function as exception-
ally effective GPx mimetics. Thus, the novel cyclic seleninate
ester 311 and spirodioxyselenurane 4,12 as well as derivatives
Substituent effects were studied in a series of aromatic cyclic
seleninate esters and spirodioxyselenuranes that function as
mimetics of the antioxidant selenoenzyme glutathione per-
oxidase. The methoxy-substituted selenurane proved the most
efficacious catalyst for the reduction of hydrogen peroxide
with benzyl thiol, and the reaction rates were enhanced for
both classes by electron-donating substituents. Hammett plots
indicated F ) -0.45 and -3.1 for the seleninates and
selenuranes, respectively, suggesting that oxidation at Se is
the rate-determining step in their catalytic cycles.
(5) (a) Ganther, H. E. Chem. Scr. 1975, 8a, 79. (b) Ganther, H. E.; Kraus,
R. J. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic
Press: New York, 1984; Vol. 107, pp 593-602. (c) Stadtman, T. C. J. Biol.
Chem. 1991, 266, 16257. (d) Tappel, A. L. Curr. Top. Cell Regul. 1984, 24, 87.
(e) Flohe´, L. Curr. Top Cell Regul. 1985, 27, 473.
Aerobic metabolism produces peroxides, other reduced
oxygen species, and free radicals that contribute to oxidative
stress in living organisms. This has been implicated in a variety
of degenerative processes and disease states in human patients,
including inflammation, cardiovascular disease, mutagenesis and
cancer, dementia, and possibly even aging.1,2 These deleterious
effects are mitigated by a variety of dietary antioxidants,1b,3,4
as well as by several endogenous enzymes that catalyze the
destruction of peroxides and other harmful reduced oxygen
(6) The structure of GPx is tetrameric, with a selenocysteine residue in each
of the four subunits: Epp, O.; Ladenstein, R.; Wendel, A Eur. J. Biochem. 1983,
133, 51.
(7) For reviews, see: (a) Mugesh, G.; du Mont, W.-W.; Sies, H. Chem. ReV.
2001, 101, 2125. (b) Mugesh, G.; Singh, H. B. Chem. Soc. ReV. 2000, 29, 347.
(c) Mugesh, G.; du Mont, W.-W. Chem. Eur. J. 2001, 7, 1365.
(8) (a) Mu¨ller, A.; Cadenas, E.; Graf, P.; Sies, H. Biochem. Pharmacol. 1984,
33, 3235. (b) Wendel, A.; Fausel, M.; Safayhi, H.; Tiegs, G.; Otter, R. Biochem.
Pharmacol. 1984, 33, 3241. (c) Parnham, M. J.; Kindt, S. Biochem. Pharmacol.
1984, 33, 3247. (d) Mu¨ller, A.; Gabriel, H.; Sies, H. Biochem. Pharmacol. 1985,
34, 1185. (e) Safayhi, H.; Tiegs, G.; Wendel, A. Biochem. Pharmacol. 1985,
34, 2691. (f) Wendel, A.; Tiegs, G. Biochem. Pharmacol. 1986, 35, 2115. (g)
Fischer, H.; Dereu, N. Bull. Soc. Chim. Belg. 1987, 96, 757. (h) Haenen,
G. R. M. M.; De Rooij, B. M.; Vermeulen, N. P. E.; Bast, A. Mol. Pharmacol.
1990, 37, 412. (i) Glass, R. S.; Farooqui, F.; Sabahi, M.; Ehler, K. W. J. Org.
Chem. 1989, 54, 1092.
(1) (a) OxidatiVe Stress; Sies, H., Ed; Academic Press: London, 1985. (b)
Free Radicals and OxidatiVe Stress: EnVironment, Drugs and Food AdditiVes;
Rice-Evans, C., Halliwell, B., Lunt, G. G., Eds.; Portland Press: London, 1995.
(2) (a) Free Radicals in Biology; Pryor, W. A., Ed.; Academic Press: New
York, 1976-1982; Vols. 1-5. (b) Free Radicals in Molecular Biology, Aging
and Disease; Armstrong, D., Sohal, R. S., Cutler, R. G., Slater, T. F., Eds.;
Raven Press: New York, 1984.
(3) OxidatiVe Processes and Antioxidants; Paoletti, R., Samuelsson, B.,
Catapano, A. L., Poli, A., Rinetti, M., Eds.; Raven Press: New York, 1994.
(4) For this reason, selenium is an essential nutrient; see: (a) Selenium in
Biology and Human Health; Burk, R. F., Ed.; Springer-Verlag: New York, 1994.
(b) Shamberger, R. J. Biochemistry of Selenium; Plenum Press: New York, 1983.
(9) For a list of potential medicinal applications of ebselen, see: Fong, M. C.;
Schiesser, C. H. Tetrahedron Lett. 1995, 36, 7329.
(10) ALT 2074 was formerly known as BXT 51072. For lead references,
see: (a) Moutet, M.; D’Alessio, P.; Malette, P.; Devaux, V.; Chaudie`re, J. Free
Radical Biol. Med. 1998, 25, 270. (b) Erdelmeier, I.; Tailhan-Lomont, C.; Yadan,
J.-C. J. Org. Chem. 2000, 65, 8152. (c) Jacquemin, P. V.; Christiaens, L. E.;
Renson, M. J.; Evers, M. J.; Dereu, N. Tetrahedron Lett. 1992, 33, 3863.
4252 J. Org. Chem. 2008, 73, 4252–4255
10.1021/jo800381s CCC: $40.75 2008 American Chemical Society
Published on Web 04/24/2008

SCHEME 2
SCHEME 4
SCHEME 3
pearance of the thiol or the formation of the corresponding
disulfide (BnSSBn). The distinct methylene signals of the
benzylic compounds provide an additional means for monitoring
the reactions in deuterated solvents by NMR and for investigat-
ing intermediates. For convenience, we compared the activities
of the catalysts by means of half-lives (t1/2), representing the
time required for the conversion of 50% of the thiol to its
disulfide under the above conditions.
In general, aromatic selenium compounds tend to have lower
toxicities than their aliphatic counterparts, mainly because of
their greater resistance to metabolic degradation to highly toxic
inorganic selenium compounds.7b,17 Unfortunately, the aromatic
derivatives 7a, 8a, 9, and 10 displayed debilitated activity
relative to the aliphatic analogues 3 and 4. We now report the
effect of substituents on the aromatic compounds 7a and 8a as
part of an effort to improve their catalytic activities while
retaining the expected lower toxicity. Furthermore, these experi-
ments provided additional insight into the mechanism of the
respective catalytic cycles of cyclic seleninate esters and
spirodioxyselenuranes.
of these prototypes,13 catalyzed the reduction of tert-butyl
hydroperoxide or hydrogen peroxide, using benzyl thiol as the
sacrificial thiol. The catalytic cycles, shown in Schemes 2 and
3, respectively, proved distinct from each other and from that
of GPx, as shown in Scheme 1. Moreover, selenol and selenolate
intermediates are capable of reducing dioxygen, resulting in
the formation, as well as the destruction, of reactive oxygen
species.14 The absence of selenol intermediates from Schemes
2 and 3 is therefore a further advantage. We also reported that
5 and 6, the tellurium analogues of 3 and 4, respectively,
displayed greater catalytic activity, while the benzo derivatives
7a, 8a, 9, 10 were considerably less active.13a Compounds 7a
and 8a were also investigated by Singh and co-workers.15
Cyclic seleninate ester 7a and spirodioxyselenurane 8a were
obtained as described previously.13a The desired substituted
derivatives 7b-f and 8b-e were prepared from the correspond-
ing diselenides 11b-f, as shown in Schemes 4 and 5. The latter
were in turn obtained from 5-substituted anthranilic acids by
the same general procedure that was previously employed for
the unsubstituted diselenide 11a.13a,18 The diselenides were
treated with lithium aluminum hydride to reduce their carboxylic
acid substituents to benzylic alcohols and their diselenide
moieties to selenolates. Direct treatment of the latter with allyl
iodide afforded the allyl selenides 13b-f in poor yields. A more
efficient procedure involved the air oxidation of the selenolates
produced by the lithium aluminum hydride reduction, followed
by isolation of diselenides 12b-f. Reduction with sodium
borohydride followed by allylation provided improved yields
of 13b-f, which were then converted in one step to the products
7b-f by oxidation with excess tert-butyl hydroperoxide (Scheme
4).19 Diselenide 12g was best prepared by metalation of
bromobenzyl alcohol 14, followed by selenation of the dianion
and air oxidation. It was then converted to 7g in the usual
In order to compare the relative catalytic activities of various
GPx mimetics, we devised a simple assay11,12,16 in which the
catalyst (10 mol %), benzyl thiol (BnSH), and an excess of either
tert-butyl hydroperoxide or hydrogen peroxide are allowed to
react at 18 °C in dichloromethane-methanol solution. The
reactions are conveniently monitored by HPLC for the disap-
(11) (a) Back, T. G.; Moussa, Z. J. Am. Chem. Soc. 2002, 124, 12104. (b)
Back, T. G.; Moussa, Z. J. Am. Chem. Soc. 2003, 125, 13455.
(12) Back, T. G.; Moussa, Z.; Parvez, M. Angew. Chem., Int. Ed. 2004, 43,
1268.
(13) (a) Back, T. G.; Kuzma, D.; Parvez, M. J. Org. Chem. 2005, 70, 9230.
(b) Kuzma, D.; Parvez, M.; Back, T. G. Org. Biomol. Chem. 2007, 5, 3213. (c)
Back, T. G.; Kuzma, D.; Berkowitz, N. PCT Int. Appl. WO 2007044641, 2007.
(14) Chaudiere, J.; Courtin, O.; Leclaire, J. Arch. Biochem. Biophys. 1992,
296, 328.
(15) Tripathi, S. K.; Patel, U.; Roy, D.; Sunoj, R. B.; Singh, H. B.;
Wolmersha¨user, G.; Butcher, R. J. J. Org. Chem. 2005, 70, 9237.
(16) Back, T. G.; Dyck, B. P. J. Am. Chem. Soc. 1997, 119, 2079.
(17) Nogueira, C. W.; Zeni, G.; Rocha, J. B. T. Chem. ReV. 2004, 104, 6255.
(18) Lesser, R.; Weiss, R. Ber. Dtsch. Chem. Ges. 1913, 2640.
J. Org. Chem. Vol. 73, No. 11, 2008 4253

TABLE 1. Catalytic activities of GPx mimetics
SCHEME 5
entry
catalyst
t1/2 (h)
1
2
3
4
5
6
7
8
ebselen (1)
3
24
18a
50
72
69
62
48
57
7a
7b
7c
7d
7e
7f
9
7g
4
38
10
11
12
13
14
15
16
17
0.2a
2.9
22
8a
8b
8c
8d
8e
8f
25
31
3.6
8g
0.5
a Data taken from ref 13a using slightly different conditions.
manner. Direct oxidation of diselenides 12b-f with excess
hydrogen peroxide or tert-butyl hydroperoxide also afforded
cyclic seleninates 7b-f, but the products contained impurities
which proved difficult to remove.
Diselenides 12a-f were converted to spirodioxyselenuranes
8a-f by reduction to their selenolates, followed by treatment
with the diazonium salts generated in situ from 2-aminobenzyl
alcohols 15a-f, which were in turn obtained from the reduction
of the corresponding anthranilic acids with lithium aluminum
hydride. The resulting selenides 16a-f contained small amounts
of the corresponding diselenides and were typically oxidized
with excess hydrogen peroxide or tert-butyl hydroperoxide
without further purification to afford the desired spiro com-
pounds. Selenide 16g was best obtained by selenation of the
FIGURE 1. Hammett plot for cyclic seleninates 7a-g.
respectively) proved superior to 1, with the methoxy derivative
8g (entry 17) displaying a t1/2 48 times smaller than that of 1,
and approaching that of the aliphatic spirodioxyselenurane 4
(entry 10).
Moreover, there appears to be a clear correlation between
catalytic activity as measured by the t1/2 values in Table 1 and
the electron-donating/withdrawing nature of the substituents in
both the cyclic seleninates and spirodioxyselenuranes. Thus,
while electron-withdrawing substituents such as the halogens
in 7b-d and 8b-d (entries 4-6 and 12-14) suppressed
catalytic activity, the strongly electron-donating methoxy groups
in 7g and 8g enhanced it considerably (entries 9 and 17). The
effects of the phenyl and methyl substituents in derivatives 7e,
7f, and 8e were relatively modest (entries 7, 8, and 15). The
correlation of catalytic activity with substituent effects was also
evident in the Hammett plots for the above reactions. Thus, the
overall rates for the reactions of benzyl thiol with hydrogen
peroxide were measured in the presence of the unsubstituted
cyclic seleninate 7a and the substituted derivatives 7b-g. The
Hammett plot was made using conventional values for σpara
resulting in a reaction constant F ) -0.45 (Figure 1). The
negative slope indicates that the transition state of the rate-
determining step is stabilized by electron-donating substituents
and destabilized by electron-withdrawing groups. The similar
20
dianion of 14 with Se(dtc)2 (Scheme 5).
Substituted cyclic seleninates 7a-g and spirodioxysele-
nuranes 8a-e,g were then subjected to our assay for catalytic
activity, using benzyl thiol and hydrogen peroxide. The phenyl-
substituted spirodioxyselenurane 8f could not be purified
completely and proved too insoluble for measurement under
the conditions of the assay. The t1/2 values thus obtained are
indicated in Table 1, along with those of the aliphatic analogues
3 and 4 for comparison. A control reaction performed in the
absence of a selenium-based catalyst under the same conditions
produced a t1/2 of 176 h. These results indicate that the aromatic
cyclic seleninate esters 7a-g (entries 3-9) are all inferior to
ebselen (1; entry 1) and the aliphatic derivative 3 (entry 2) in
catalytic activity, as measured by this assay. On the other hand,
spirodioxyselenuranes 8a, 8e, and 8g (entries 11, 15 and 17,
21
,
(19) This process presumably proceeds via selenoxide formation from 13,
[2,3]sigmatropic rearrangement to the corresponding selenenate ester, followed
by further oxidation and cyclization to afford 7.
(20) Foss, O. Inorg. Synth. 1953, 4, 91.
(21) Smith, M. B.; March, J. March’s AdVanced Organic Chemistry, 5th ed.:
Wiley: New York, 2001; p 370 and references cited therein.
4254 J. Org. Chem. Vol. 73, No. 11, 2008

of thiol and formation of disulfide with time (see the Supporting
Information) also supports the oxidation of Se(II) to Se(IV) as
the rate-limiting step in both series of compounds.
Finally, it is especially noteworthy that the methoxy-
substituted spirodioxyselenurane 8g displays catalytic activity
superior to that of all of the other aromatic compounds in Table
1 and approaches that of the aliphatic prototype 4 (see entries
10 and 17 in Table 1). Thus, the exploitation of substituent
effects has enabled us to emulate the high activity of the aliphatic
species 4 while circumventing the generally greater toxicity
associated with aliphatic selenium compounds.
Experimental Section
FIGURE 2. Hammett plot for spirodioxyselenuranes 8a-e,g.
Compounds 7b-g and 8b-e,g were prepared by variations of
the procedures used earlier for the preparation of 7a and 8a,
respectively.13a Sample procedures are described in the Supporting
Information, along with characterization data for new compounds.22
Catalytic activity was measured by adding the catalyst (10 mol
%) to a mixture of 28% hydrogen peroxide (0.035 M) and redistilled
benzyl thiol (0.031 M) in dichloromethane-methanol (95:5) while
maintaining the temperature at 18 °C. The reactions were monitored
by HPLC analysis, using a UV detector at 254 nm and a reversed-
phase column (Novapak C18; 3.9 × 150 mm), with naphthalene
(0.0080 M) as an internal standard. Acetonitrile-water was
employed as the solvent (gradient: 60:40 to 80:20 over 15 min with
a flow rate of 0.9 mL/min). Each t1/2 value in Table 1 is the average
of at least two runs. Kinetic plots are provided in the Supporting
Information.
treatment of the spirodioxyselenuranes also produced a negative
slope, but of considerably greater magnitude (F ) -3.1).
We hypothesize that the mechanisms for the catalytic cycles
of the aromatic analogues 7 and 8 are similar to those reported
previously for the aliphatic compounds 3 and 4, respectively
(Schemes 2 and 3). Since the steps involving substitution or
reductive elimination by the nucleophilic thiol would be
facilitated by a more electrophilic selenium center, they would
be enhanced by electron-withdrawing substituents. On the other
hand, steps where Se(II) is oxidized to Se(IV) would be expected
to proceed more rapidly in the presence of electron-donating
substituents that are capable of stabilizing the development of
increased positive charge on the selenium atom. The negative
values of F indicate that the latter is the case, supporting the
hypothesis that the oxidation of selenium comprises the rate-
determining step in both series of compounds 7 and 8. The large
negative value of F in Figure 2 reveals that the oxidation of the
selenide to its corresponding selenoxide in Scheme 3 is
particularly sensitive to the nature of para substituents. More-
over, the kinetic plots of the assays for catalysts 8 (see the
Supporting Information) show positive y-intercepts, indicating
anomalously high rates of formation in the early stages of the
reaction. This is consistent with the rapid consumption of the
catalyst (10 mol %) and concomitant conversion of thiol to
disulfide in the first two steps in Scheme 3, prior to the rate-
determining oxidation of Se(II) to Se(IV), and eventual regen-
eration of the catalyst. The linear (zero-order) disappearance
Acknowledgment. We thank the Natural Sciences and
Engineering Research Council of Canada for financial support.
Supporting Information Available: Typical procedures,
characterization of new compounds, including 1H and 13C NMR
spectra, and kinetic plots for the results summarized in Table
1. This material is available free of charge via the Internet at
http://pubs.acs.org.
JO800381S
(22) Among compounds 7, 8, 11-13, and 16, only the unsubstituted
derivatives (series a in Table 1) and diselenide 11d have been reported previously.
For the unsubstituted compounds, see ref 13a; for 11d, see: Vafai, M; Renson,
M. Bull. Soc. Chim. Belg. 1966, 75, 145.
J. Org. Chem. Vol. 73, No. 11, 2008 4255